Project Summary/Abstract - Core 3 ? Specialized Macromolecular Crystallography This Technology Operations Core will serve up nine advanced technologies that are seriously needed by members of the structural biology user community working on particularly challenging problems. These are: 1) multi-crystal strategy for when one crystal is not enough, 2) native element phasing for when preparing derivatives is impractical, 3) in-situ diffraction from trays for when the crystals are too fragile to handle, 4) diffraction at non-cryogenic temperatures, for functional studies or when cryo-protection makes diffraction worse, 5) alternative visualization technologies for finding crystals in loops ranging from polarization microscopy to online X-ray tomography (CBOXAR) and raster grid searches of a small x-ray beam over the face of the sample to probe for diffraction quality exhaustively, 6) data quality prediction based on first- principles and at-scale diffraction simulation technology to deduce the best possible data collection parameters based on all available information about a given sample, 7) a comprehensive array of available beam properties, including our soon-to-be-completed micro-focus GEMINI beamline 8) automatic optical re-centering technology (AUORA) to enable autonomous migration of experiments to any ALS beamline, for optimizing beamline utilization 9) a clear ?targeting file format? specification so that, if necessary, experiments can be migrated outside the ALS, such as to X-ray Free Electron Lasers. All these technologies will be tied together by the ALS-ENABLE website, which will track not just the samples and data processing results, but the inter-compatibility relationships between them. This will be essential for managing the combinatorial explosion of data sets that must be explored to stitch together the best possible complete data set for a given project. This averaging will be key to native-element phasing, where the signal from any single sample is seldom good enough for phasing. The website will also serve as a knowledge base, capable of making recommendations to Users based on all the data they currently have, and the predictions provided by our uniquely accurate diffraction simulation technology. For example, it will be recommended that they try in-situ diffraction if SAXS (TOC 2) shows that their molecule is intrinsically ordered but diffraction is stuck at 6 resolution. If in-situ diffraction is also poor, then the recommendation will be to search for a new crystal form using X-ray Footprinting (outside ALS-ENABLE) or cryo-EM (outside ALS). We expect this Resource will appeal to a wide regional and national geographic distribution of users. By addressing the problem of poor diffraction, the need for functional studies at multiple temperatures, the need for native element phasing, and by de-centralizing the crystal centering problem we will leverage the diversity of the ALS beamlines into a coherent and easily accessible Resource.